Biological Study And RNA Aptamer Selection And Identification Of Hepatic Asialoglycoprotein Receptor

Objective1. To study the occurrence mechanism, expression level and protein characteristics of the major subunit variants of human asialoglycoprotein receptor, and lay a foundation for further study of the function of ASGPR and for hepatic targeting therapy through ASGPR.2. To obtain RNA aptamer with high affinity and specificity to the human liver specific asialoglycoprotein receptor (ASGPR) and lay a foundation for developing new reagents or drugs for the diagnosis and the targeting treatment of liver diseases.Methods1. Two major subunit variants of ASGPR (H1a and H1b) were cloned from normal human liver tissues and HepG2 cells, the occurrence mechanism of variant H1b was analyzed by sequences comparison. The expression levels of H1a and H1b in normal liver tissues and HepG2 cells were determined by real-time fluorescence quantitative PCR, as well as the level changes in HBV or HCV infected cells and HCC tissues.2. H1b specific peptide was synthesized and coupled with keyhole limpet hemocyanin (KLH) for immunization. Then H1b-KLH conjugation was injected into mouse subcutaneously to produce polyclonal antibody. The titer and specificity of the antibody were confirmed. Soluble ASGPR (sASGPR) in normal human sera or HepG2 cell supernatants were purified by lactose-agarose affinity chromatography, and the compositions of sASGPR were analyed by specific antibodies. The expression and location of protein H1b in liver tissues were also determined by immunohistochemistry. 3. A single-stranded 115 nucleotides (nt) random DNA library containing 25 random oligonucleotides was synthesized in vitro, then random RNA aptamers library was constructed by in vitro transcription. Aptamers that can specifically bind to human hepatic asialoglycoprotein receptor was isolated from the RNA random library by using the SELEX (systematic evolution of ligands by exponential enrichment) procedure. Selected aptamers were analyzed by sequencing, and the secondary structures of these aptamers were prediced and analyzed by using RNA Structure Program.4. Aptamers were labeled by ³²P, then filter biding assay and gel shift assay were performed to determine the specificity and affinity of the selected aptamers. One apatamer was also labeled by FITC, and the binding between the aptamer and the human hepatoma cell lines HepG2 and HuH-7 were identified by fluorescent staining.Results1. Two variants of ASGPR H1, designated H1a and H1b, are extensively expressed in both human liver tissues and in human hepatoma cell lines HepG2 and Huh7. Cloning of ASGPR H1 splice variant cDNAs and subsequent sequence analysis revealed that they differ only in the presence of 117 nt. This 117 nt segment corresponds to exon 2 in the genomic sequence of ASGPR, and the typical AG/GT 5' consensus splice donor and acceptor sites were found at each end of the 117 nt segment, strongly suggesting that ASGPR H1b is generated by alternative splicing of ASGPR mRNA.2. The ratio of ASGPR H1a to H1b was about 2.6:1 in normal human liver tissue and 5.2:1 in HepG2 cells. H1b expression were decreased by approximately 60% in HBV or HCV infected cells, and were decreased more than 90% in HCC tissues.3. Both the H1b protein and a functional soluble ASGPR (sASGPR) composed of H1b and H2 were identified in both normal human sera and in the supernatant of HepG2 cells. Immunohistochemistry analysis shows that protein H1b in liver tissues can not accumulate at the cellular membrane, but in the cytoplasm.4. Twelve rounds of selection and amplification were performed, and the aptamers which can bind to H1 were significantly enriched. Round 12 RNAs were amplified by RT-PCR and cloned. Forty-eight randomly picked plasmid clones were sequenced. The secondary structures of these aptamers were predicted with RNA Structure Program. Most aptamers of Round 12 RNAs were represented by only two kinds of structures, and their percentages of composition of Round 12 RNAs were 45.8% and 33.3%, respectively. One aptamer, H1-A25, was found to have high affinity to protein H1. the dissociation constant for the aptamer:H1 complex was estimated to be 48.79 nM.5. While P³²-labeled H1-A25 significantly bound to H1 protein on the nitrocellulose filter (determined by nitrocellulose filter binding assay), its binding to HBsAg, the HBV S antigen that served as a negative control, was similar to that of probe alone. Furthermore, gel mobility shift assays also confirmed the specific binding of aptamer H1-A25 to H1 protein, as only recombinant H1 but not HBsAg, could cause the retardation of mobility of P³²-labeled H1-A25 probe. The addition of 100-fold excess unlabeled aptamer to the reaction system abolished the aptamer-H1 interaction.6. FITC labeling H1-A25 specifically bound to the ASGPR-expressing hepatoma cell lines HepG2 and Huh-7. However, no binding was shown to the ASGPR-negative cell line HeLa. The fluorescence signal was significantly reduced by preincubating HepG2 cells with anti-H1 polyclonal antibody and can be nearly blocked by adding 100-fold excess unlabeled H1-A25.Conclusions1. Two variants of ASGPR H1, designated H1a and H1b, are extensively expressed in both human liver tissues and in human hepatoma cell lines HepG2 and Huh7.2. The protein encoded by the H1b variant is a secreted form of H1.3. sASGPR is a hetero-oligomeric complex of the secreted form of H1 and H2 and is able to bind a lactose-agarose substrate.4. RNA aptamer H1-A25 that can specifically bind to human hepatic ASGPR with high affinity was successfully selected by using SELEX.5. Aptamer H1-A25 can specifically bind to human hapatocytes.Significances of the study1. Our works provide the first evidence of the existence of splice variant H1b of human hepatic ASGPR H1, and prove that the protein encoded by the variant H1b is a secreted form of H1 which participates in the composing of sASGPR complex. The study improved the understanding of sASGPR, and laid a foundation for further comprehensive study of the function of ASGPR.2. This is the first reported RNA aptamer which could bind to a human hepatic specific receptor. And this newly isolated aptamer could be modified to deliver imaging, diagnostic, and therapeutic agents targeted at hepatic parenchymal cells.